Deletion of a prophage-like element causes attenuation of Salmonella enterica serovar Enteritidis and promotes protective immunity

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a wide host range serovar belonging to the S. enterica genus. Worldwide, it is one of the most frequent causes of food borne disease. Similar to S. Typhimurium, some virulence genes of S. Enteritidis are located in pathogenicity islands and prophages. In this study we have generated a mutant strain of S. Enteritidis lacking a prophage-like element, denominated ?SE12. The resulting mutant strain was attenuated and promoted protective immunity in infected mice. Although S. Enteritidis strains lacking the complete prophage ?SE12 remained capable of surviving inside phagocytic cells, they showed a significantly reduced capacity to colonize internal organs and failed to cause lethal disease in mice. Consistent with these data, infection with S. Enteritidis strains lacking prophage ?SE12 promoted the production of anti-Salmonella IgG antibodies and led to protection against a challenge with virulent strains of S. Enteritidis. These results suggest that strains lacking this prophage can induce a protective immunity in mice and be considered as potential attenuated vaccines against S. Enteritidis.     

Population structure, origins and evolution of major Salmonella enterica clones

The genus Salmonella consists of two species S. enterica and S. bongori. S. enterica has a well defined subspecies structure with seven subspecies consistently delineated by sequence variation. Frequency of recombination between subspecies and within a subspecies is markedly different. Subspecies I undergoes frequent recombination as demonstrated recently, demystifying the long-held belief that Salmonella is a highly clonal organism. The majority of disease causing serovars are from subspecies I with the most important serovars in human health being Typhimurium and Typhi. Typhimurium has developed considerable diversity and may be a very old serovar. The majority of the isolates belong to a single clonal complex by multilocus sequence typing. Typhimurium isolates are divided into phage types and some of the phage types do not have a single origin as determined using mutational changes. Phage type DT104 is heterogeneous and represented in multiple sequence types, with its multidrug-resistant variant most successful causing epidemics in many parts of the world. Typhi, a human restricted serovar, is relatively young compared to Typhimurium, and has a low level of sequence variation. Single nucleotide polymorphisms (SNPs) have been shown to be very useful for typing and resolving relationships within Typhi. Genome sequences of 19 isolates revealed more than 1700 SNPs. The fully resolved phylogenetic tree allows one to trace the mutational changes occurred during clonal diversification. Genome wide SNPs have greatly enhanced our understanding of the evolution of Salmonella clones.     

Bidirectional Salmonella enterica serovar Typhimurium transfer between bare/glove hands and green bell pepper and its interruption

The aim of this study was to quantify the amount of Salmonella enterica serovar Typhimurium transferred from volunteers' hands (bare or gloved) to green bell peppers and vice versa; and to assess the effectiveness of hand hygiene techniques. The highest and lowest percentages of bacterial transfer were achieve from green bell peppers to gloved hands (46.56%) and from bare hands to green bell peppers (0.21%), respectively. The highest and lowest Log10 reductions of S. Typhimurium were achieved by the combination of hand washing and alcohol-based gel (4.38 Log10) and iodine solution (2.08 Log10), respectively. This study provides important information concerning the transfer's efficiency of S. Typhimurium from hands to fresh produce and from fresh produce to hands. The study also showed that gloved hands, could be a mean of transfer of S. Typhimurium between green peppers and hands, and the best hand hygiene technique was the combination of hand washing and alcohol-based gel.     

重组人乳铁蛋白对小鼠沙门菌感染抑制效果

目的研究转基因水稻来源的重组人乳铁蛋白(rhLF)在小鼠体内抑制沙门菌感染的效果。方法选择Balb/c小鼠建立沙门菌感染模型; 将Balb/c小鼠按体重随机分为2组,即沙门菌对照组和rhLF干预组,每组12只;染菌前2h,rhLF干预组小鼠经口灌胃20 mg/mL rhLF 0.3 mL,对照组小鼠经口灌胃0.3 mL PBS;之后每天灌胃相同量的rhLF(6 mg/d)或PBS;每天观察小鼠一般情况;7 d后,心脏穿刺取小鼠血液观察菌血症,取肝脏和脾脏组织进行细菌计数分析,取肝脏、脾脏、肾脏和小肠进行病理观察。结果各组小鼠体重变化、生存情况在组间无明显差异(P>0.05);2组小鼠的肝脏重和肝体比值、脾脏重和脾体比值相似(P>0.05);rhLF干预组小鼠的血液、肝脏和脾脏的染菌率和染菌量与对照组相比,呈现降低趋势,但差异无统计学意义(P>0.05);病理分析显示rhLF干预组小鼠的炎症程度有减轻,但差异无统计学意义(P>0.05)。结论6 mg/d的rhLF不能明显减轻小鼠沙门菌感染的炎症程度。     

Attenuated Salmonella enterica serovar Typhimurium lacking the ZnuABC transporter: An efficacious orally-administered mucosal vaccine against salmonellosis in pigs

We have recently demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium unable to synthesize the zinc transporter ZnuABC (S. Typhimurium ΔznuABC), is able to protect mice against systemic and enteric salmonellosis and is safe in pigs. Here, we have tested the protective effects of S. Typhimurium ΔznuABC in pigs. Resistance to challenge with the fully virulent strain S. Typhimurium ATCC 14028 was assessed in animals vaccinated with S. Typhimurium ΔznuABC (two dosages tested), in controls vaccinated with a formalin-inactivated virulent strain and in unvaccinated controls. Clinical signs of salmonellosis, faecal shedding and bacterial colonization of organs were used to assess vaccine-induced protection. After the challenge, pigs vaccinated with the attenuated S. Typhimurium ΔznuABC strain did not display clinical signs of salmonellosis (fever or diarrhoea). The vaccine also reduced intestinal tract colonization and faecal shedding of the fully virulent Salmonella strain, as compared to control groups. S. Typhimurium ΔznuABC represents a promising candidate vaccine against salmonellosis in pigs.     

江苏省2012-2015年伤寒/副伤寒沙门菌耐药及分子分型研究

目的 回顾性分析2012-2015年江苏省伤寒/副伤寒沙门菌的耐药表型和分子分型特征。方法 采集江苏省伤寒/副伤寒监测病例的血液和粪便标本,分离培养伤寒/副伤寒沙门菌后进行生化鉴定和血清分型;采用Kirby-Bauer纸片法检测菌株对抗菌药物的敏感性;应用PFGE技术对伤寒和甲型副伤寒沙门菌进行分子分型。结果 134株伤寒沙门菌和甲型副伤寒沙门菌对萘啶酸的耐药率最高,分别为61.2%和86.7%,其余抗生素的耐药率均低于15.0%。伤寒和甲型副伤寒沙门菌中只耐1种抗生素的菌株最多,多重耐药菌在伤寒沙门菌中占2.6%,而在甲型副伤寒沙门菌中占13.3%。2015年伤寒沙门菌的全敏感菌株构成比较2012年增加了44.3%,同年副伤寒沙门菌中出现了耐5种和6种抗生素的多重耐药株。甲型副伤寒沙门菌可分为8种PFGE型别,多重耐药株与其他菌株条带相似性低,不同型别与耐药谱有对应关系;伤寒沙门菌可分为68种PFGE型别,不同型别间变异度较大,与耐药谱无对应关系。结论 2012-2015年江苏省伤寒/副伤寒沙门菌对抗生素的耐药性总体随时间降低,耐药种类数有所增加。伤寒沙门菌株PFGE带型呈现多样性,无耐药谱对应性;而甲型副伤寒菌株PFGE带型较少,有耐药谱对应性。需要加强重点地区重点型别菌株的监测。     

Oral delivery of a DNA vaccine against tuberculosis using operator-repressor titration in a Salmonella enterica vector

Attenuated Salmonella enterica offers a vaccine delivery route that has the benefits of enhanced immunogenicity and oral delivery. The majority of immunization studies have been conducted to deliver recombinant proteins, expressed from a gene that is either chromosomally integrated or carried on a low- or medium-copy number plasmid. There are, however, an increasing number of reports demonstrating the delivery of DNA vaccines, but the high-copy number plasmids that are preferentially used for this application are unstable in Salmonella. Here, we use the Operator-Repressor Titration (ORT) plasmid maintenance system in Salmonella enterica serovar Typhimurium to deliver a high-copy number plasmid expressing the Mycobacterium tuberculosis gene mpt64 to mice. MPT64 expression was detected in phagocytes using immunofluorescence microscopy following Salmonella-mediated delivery of the DNA vaccine. The indicative CD8+ responses measured by antigen-specific IFN-γ were higher from the live bacterial vector than from injected plasmid DNA, and a reduction in the pulmonary bacterial load was seen following an aerogenic challenge. This illustrates the potential of live attenuated Salmonella as oral tuberculosis vaccine vectors.     

河南省2009-2011年伤寒副伤寒沙门菌分子分型与耐药研究

目的 分析2009-2011年河南省伤寒副伤寒沙门菌临床分离株耐药状况与PFGE分型特征。方法 根据PulseNet公布的伤寒副伤寒沙门菌PFGE分型操作规程与美国临床实验室标准协会(CLSI)沙门菌K-B法药敏测试方案,对2009-2011年分离自河南省监测哨点医院78株伤寒副伤寒沙门菌进行抗生素药敏测试与PFGE分子分型。结果 78株伤寒副伤寒沙门菌对8类13种抗生素均有不同程度的耐药,62株为多重耐药菌株(79.5%),其中耐2～3种4株(5.1%),耐5～8种41株(52.6%),耐9～10种14株(17.9%),耐11～12种3株(3.8%);菌株对头孢类、喹诺酮类等5类抗生素耐药率总体呈上升趋势。经XbaⅠ酶切与PFGE后,获得14种带型,每种带型包含菌株数1～47株不等,相似度为66.03%～100.00%。结论 2000-2011年河南省临床分离的伤寒副伤寒沙门菌耐药状况普遍比较严重,PFGE带型呈现多样性的同时又具有较显著的优势带型特点,部分带型与其对应的耐药谱具有一定的关联性和聚集性。     

sopB of Salmonella enterica serovar Typhimurium is a potential DNA vaccine candidate in conjugation with live attenuated bacteria

The immune response against Salmonella is multi-faceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify a protective T cell epitope(s) of Salmonella, as cell mediated immunity conferred by CD8+ T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the genbank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of Salmonella enterica serovar Typhimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi). They were subjected to BIMAS and SYFPEITHI analysis to map MHC-I and MHC-II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire SopB and SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5-fold on day 4 and day 8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone.     

Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium

Protective immune responses in shrimp induced by DNA vaccines against white spot syndrome virus (WSSV) with intramuscular injection have been reported in recent reports. In this study, we investigated the utilities of attenuated Salmonella enterica serovar Typhimurium (Salmonella typhimurium) as a bactofection vehicle for the oral delivery of a DNA vaccine plasmid to crayfish (Cambarus clarkii). The DNA vaccine plasmid pcDNA3.1-VP28, encoding viral envelope protein VP28, was transformed to an attenuated S. typhimurium strain SV4089 and the resulting recombinant bacteria named SV/pcDNA3.1-VP28 were used to orally immunize crayfish with coated feed. Successful delivery of the DNA vaccine plasmid was shown by the isolation of recombinant bacteria SV/pcDNA3.1-VP28 from the vaccinated crayfish. The distribution analysis of plasmid pcDNA3.1-VP28 in different tissues revealed the effective release of DNA vaccine plasmid into crayfish. RT-PCR and immunoflurescence results confirmed the expression of protein VP28 in the vaccinated crayfish. Challenge experiments with WSSV at 7, 15, 25 days post-vaccination demonstrated significant protection in immunized crayfish with relative survival rate 83.3%, 66.7% and 56.7%, respectively. Studies on stability and safety of SV/pcDNA3.1-VP28 showed the recombinant bacteria could exist in crayfish at least 7 days but not more than 10 days and without any observable harm to the host. Our study here demonstrates, for the first time, the ability of attenuated Salmonella as a live vector to orally deliver a DNA vaccine against WSSV into the arthropod crayfish and provides a new way to design more practical strategies for the control of WSSV and other invertebrate pathogens.     

Salmonella enterica serovar Typhimurium ruvB mutant can confer protection against salmonellosis in mice

Salmonella enterica is an important pathogen that causes a variety of infectious diseases in animals and humans. Live attenuated vaccines generally confer better protection than killed or subunit vaccines; however, the former are limited by their inherent toxicity. We evaluated the potential of a novel candidate Salmonella vaccine strain that lacks the ruvB gene. The ruvB gene encodes a Holliday junction helicase that is required to resolve junctions that arise during the repair of non-arresting lesions after DNA replication. The deletion of this gene in Salmonella significantly impaired cell survival and proliferation within epithelial cells and macrophages. The defective virulence in ruvB mutant may be partially due to decreased expression of ssaG, a Salmonella pathogenicity island-2 gene, and increased sensitivity to hydrogen peroxide in the lack of ruvB gene. The virulence of the ruvB-deleted mutant was also greatly attenuated in BALB/c mice. The ruvB mutant conferred strong and durable immune-based protection against a challenge with a lethal dose of a virulent strain of Salmonella Typhimurium. Moreover, protective immunity was induced by a single dose of the vaccine, and the efficacy of protection was maintained for at least 6 months. These results suggest the use of the S. Typhimurium ruvB mutant as a novel vaccine.     

Protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine when delivered in-water to sheep challenged with Salmonella enterica serovar Typhimurium

Stimulation of acquired immunity to Salmonella in livestock is not feasible in neonates (which can be infected within 24 h of birth) and is challenging in feedlots, which typically source animals from diverse locations and vendors. Induction of innate immune mechanisms through mass vaccination of animals upon arrival to feedlots is an alternative approach. Transport, environmental conditions, changes in social grouping, and further handling during feedlot assembly are significant stressors. These factors, as well as concurrent exposure to a diversity of pathogens, contribute to the risk of disease. We have shown that oral immunization of calves with a modified live Salmonella enterica serovar Typhimurium vaccine strain, which lacks the DNA adenine methylase gene (S. Typhimurium dam), attenuates the severity of clinical disease, reduces fecal shedding, and promotes clearance of salmonellae following virulent homologous and heterologous challenge. This study examines the safety and efficacy of a S. Typhimurium dam vaccine in sheep via oral delivery in drinking water (ad libitum), as a means to effectively vaccinate large groups of animals. Adult merino sheep were vaccinated in drinking water −28 days, −7 days and 24 h pre and 24 h post-virulent Salmonella Typhimurium challenge which was administered via the oral route. Significant attenuation of clinical disease (temperature, appetite, and attitude) and reduction in mortality and virulent Salmonella Typhimurium fecal shedding and tissue colonization was observed in animals that received the vaccine 28 and 7 days pre-challenge. Further, vaccination did not pose a risk to stock previously infected with virulent salmonellae as mortalities and clinical disease in sheep vaccinated prior to or following virulent challenge did not differ significantly from the non-vaccinated controls. The capacity of S. Typhimurium dam vaccines delivered in drinking water to protect livestock from virulent Salmonella challenge offers an effective, economical, stressor free Salmonella prophylaxis for intensive livestock production systems.     

Impaired antibody response after immunization of HIV-infected individuals with the polysaccharide vaccine against Salmonella typhi (Typhim-Vi®)

Infections with Salmonella species, including Salmonella typhi, are more frequently observed in HIV-infected individuals than in healthy individuals. HIV-infected individuals were vaccinated with polysaccharide vaccine against Salmonella typhi (Typhim-Vi®) which is assumed to be a T-cell-independent antigen. We found that the antibody response in patients with <200×10⁶/l CD4⁺ T lymphocytes was significantly lower compared with patients with ≥200×10⁶/l CD4⁺ T lymphocytes and healthy controls. The antibody response after vaccination with the polysaccharide salmonella Vi-antigen was correlated with the number of CD4⁺ T lymphocytes and therefore Typhim-Vi can be considered to be a T-cell-independent type 2 antigen. The results of this study indicate that after vaccination the proportion of HIV-infected individuals with protective antibody concentrations against Salmonella typhi will be lower than in healthy controls.     

Immune response and protective efficacy of live attenuated Salmonella vaccine expressing antigens of Mycobacterium avium subsp. paratuberculosis against challenge in mice

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants that leads to diarrhea and eventually death. Existing vaccines have proven useful in limiting disease progression but have not been effective in preventing infection. To address this problem we constructed an attenuated Salmonella (ΔyejE; ΔssaV) strain harboring a plasmid that expressed a fusion protein comprised of the Salmonella Type III secretion system (T3SS) effector SopE and MAP antigens (85A, 85B, SOD, 74F) and evaluated its potential as vaccine candidate against MAP infection in mice. Of various SopE-MAP fusion proteins analyzed, only SopE104-Ag85A C-terminal^202–347-SOD N-terminal^1–72-Ag85B C-terminal^173–330 and SopE104-74F^{1–148 + 669–786}were successfully expressed and secreted into culture media as revealed by western blot analysis. Mice immunized with attenuated Salmonella (ΔyejE; ΔssaV) harboring the SopE104-Ag85A C-terminal^202–347-SOD N-terminal^1–72-Ag85B C-terminal^173–330 and SopE104-74F^{1–148 + 669–786}plasmid generated a potent and long lasting Th1 response characterized by production of IFN-γ. The cytokine profile varied at various time points after immunization and challenge, which showed down regulation of Th2 cytokines (IL-4, IL-10) and up-regulation of proinflammatory cytokines (IL-12 and IL-17). Further, the immune response correlated with protection as revealed by reduced bacterial load and improved histopathology of spleen and liver, which showed fewer granulomas and lower numbers of acid-fast bacilli as compared to PBS controls. Interestingly, vaccination with antigens mixed with Ribi adjuvant (Agmix + Ribi) imparted better protection than the attenuated salmonella vectored vaccine. Thus, priming with a live recombinant Salmonella strain that secretes MAP antigens represents a promising approach that could lead to development of an efficacious and cost effective vaccine for Johne's disease.     

Extended-Spectrum Cephalosporin-Resistant Salmonella enterica serovar Heidelberg Strains,the Netherlands

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains (JF6X01.0022/XbaI.0251, JF6X01.0326/XbaI.1966, JF6X01.0258/XbaI.1968, and JF6X01.0045/XbaI.1970) have been identified in the United States with pulsed-field gel electrophoresis. Our examination of isolates showed introduction of these strains in the Netherlands and highlight the need for active surveillance and intervention strategies by public health organizations.     

An outbreak of extended-spectrum, β-lactamase-producing Salmonella senftenberg in a burns ward

A strain of Salmonella senftenberg resistant to ceftazidime, gentamicin, chloramphenicol and ciprofloxacin was isolated from burn wounds of eight patients on a burns ward of a hospital in Dehli, India. The organism, which had probably been spread from patient to patient on staff hands, produced the extended-spectrum β-lactamase SHV-5 and the aminoglycoside-modifying enzymes AAC(3)II + AAC(6′). The strain was not isolated from stool cultures of any of the patients or staff, apart from the index patient who had a history of diarrhoea and fever before admission. The outbreak ended in three weeks, after the implementation of strict handwashing. This is the first report of SHV-5 β-lactamase in Salmonella spp. and also the first report of SHV-5 in India. The extended-spectrum β-lactamases that have been reported in Salmonella spp. now include the Group 2be enzymes SHV-2, SHV-5, TEM-3, TEM-25, TEM-27, CTX-M2, PER-1 and PER-2, and the Group 1 enzymes DHA-1 and CMY-2. The types of extended-spectrum β-lactamases produced by salmonellas, their association with aminoglycoside resistance and their geographical distribution are now similar to those seen in klebsiella. Increasing antibiotic resistance in these organisms is reducing therapeutic options for the treatment of invasive disease.     

Ciprofloxacin treatment failure in typhoid fever case, Pakistan

We report a case of ciprofloxacin treatment failure in a typhoid fever patient at a tertiary care hospital in Rawalpindi, Pakistan. This case shows not only the emergence of fluoroquinolone resistance in typhoid salmonellae but also the inadequacy of the current laboratory guidelines for detection of this resistance.     

Potential international spread of multidrug-resistant invasive Salmonella enterica serovar enteritidis

In developing countries, Salmonella enterica serovar Enteritidis causes substantial illness and death, and drug resistance is increasing. Isolates from the United Kingdom containing virulence-resistance plasmids were characterized. They mainly caused invasive infections in adults linked to Africa. The common features in isolates from these continents indicate the role of human travel in their spread.     

一起鼠伤寒沙门氏菌食物中毒的调查报告

1987年8月下旬福建省永安市突然发生大批发热、腹泻、腹痛病人,5天内住院1 113人。经调查,患者均主诉有食冷饮史,从患者粪便检出鼠伤寒沙门氏菌,血中检出相应抗体,并在冰淇淋、冰砖、鸡蛋和生产冷饮工具中检出同种菌,证明是因鼠伤寒菌污染冷饮品而造成的一起食物中毒。四个月后对患者、健康人、禽类、蛋品及污水进行复查,结果仍从5%污水中检出鼠伤寒菌。表明当地仍存在隐患。